The long-term objective of our proposed research is to develop the molecular basis for a Plasmodium vivax blood stage vaccine. We have identified over a dozen P. vivax proteins that by virtue of their location around the surface of the merozoite, at its apical pole, or in micronemes or rhoptries, are potential vaccine candidates. We are proposing studies for the further evaluation of six of these antigens. These include merozoite surface proteins, and reticulocyte-binding proteins. These genes encoding each of these proteins have been cloned and, as a group, determination of their sequences is nearing completion. we have begun to study the antigenicity and potential immunogenicity of each of these proteins, by expressing proteins of them in bacterial expression systems. This initial approach has been valuable for obtaining recombinant proteins that are antigenic in mice and rabbits, and elicit antibodies that recognize the native P. vivax proteins. We propose to continue using bacterial expression systems, and, in addition, to establish alternate expression systems to evaluate and compare the immunogenicity of a variety of recombinants. The Baculovirus expression system will be one such primary alternative. Recombinant proteins will be analyzed and purified for immunization of rabbits and mice for the production of antisera and monoclonal antibodies. The resulting antibody reagents will be tested first for their reactivity with native protein by immunofluorescence, immunoprecipitation, and western immunoblot analyses, and secondly for inhibitory activity preventing merozoite invasion of target erythrocytes in vitro. Recombinant proteins, which elicit antibodies that inhibit erythrocyte invasion of homologous and heterologous merozoites in vitro, will be tested in the future in appropriate monkey models. Concurrent investigations will entail the examination of selected regions of the PvMSP or PvRBP genes and proteins from P. vivax field isolates. These diversity studies will be designed to identify polymorphic regions that could potentially affect the value of selected vaccine immunogens in natural endemic settings. On the basis of the data generated in these preliminary investigation, we will evaluate which recombinants may be suitable and most promising for primate and clinical vaccine trials.